Unpredictable Sound Stress Induces a Novel Form of Hyperalgesic Priming

Abstract

Unpredictable stress induces long-lasting nociceptor plasticity, enhancing and prolonging pain, contributing to chronic pain. To enhance our understanding of how unpredictable stress induces pronociceptive neuroplasticity, we investigated its effect on nociceptor function, testing the hypothesis that unpredictable sound stress (SS) induces hyperalgesic priming, a model of acute to chronic pain transition. Male rats were subjected to intense amplitude (20-110 dB) unpredictable sound, 5- or 10-second tones presented every minute, at random times, for 30 minutes, on days 1, 3, and 4. On day 18, prostaglandin E₂ (PGE₂) was administered intradermally. In both sham and SS groups PGE₂ induces hyperalgesia, measured at 30 min post-injection. However, only in the SS group was PGE₂ hyperalgesia still present at 4 hours, indicative of hyperalgesic priming. Agents known to inhibit the expression and maintenance of Type I priming, inhibitors of protein kinase C epsilon, and peripheral protein translation, respectively, did not affect the SS-induced prolongation of PGE₂-induced hyperalgesia. In contrast, a protein kinase A (PKA) inhibitor attenuated the prolongation of PGE₂-induced hyperalgesia. This inhibition remained, unattenuated, when evaluated one month after administration of the PKA inhibitor, compatible with PKA contributing to both expression and maintenance of SS-induced hyperalgesic priming. The effect of combined Src and mitogen-activated protein kinase inhibitors, which fully reverses Type II priming, also inhibited SS-induced priming; however, this effect diminished over time. Our findings support a novel form of hyperalgesic priming induced by SS. Insights into the mechanisms underlying SS-induced priming could inform targeted interventions for pain syndromes influenced by stress.

Introduction

Chronic pain, a major public health problem affecting 60 million individuals in the US alone (24.3% of adults [42]), is characterized by a complex interplay of biological and psychological factors, with stress emerging as an important risk factor [52; 67]. Unpredictable stress has been recognized as a crucial trigger for long-lasting adaptations in the nervous system, resulting in sensitized nociceptors and an increased risk of developing chronic pain [36; 62; 63]. Thus, understanding the mechanism through which stress promotes chronic pain is an essential step in the development of effective treatment strategies to prevent and treat these pain syndromes.

Stress is a prominent risk factor for diverse chronic pain conditions such as fibromyalgia syndrome (FMS), irritable bowel syndrome (IBS) and post-traumatic stress disorder (PTSD). For example, the well-documented occurrence of chronic pain in PTSD is thought to be driven by unpredictable stress- and trauma-induced alterations in neuroendocrine stress axes, and structural changes in neural pathways responsible for fear, pain processing, and emotional regulation [14; 43; 59]. Up to 50% of individuals with PTSD experience chronic pain [58]. Furthermore, patients with both chronic pain and a diagnosis of PTSD demonstrate worse functional status, greater distress, and poorer responses to therapeutic interventions [49] .

Inflammation, peripheral neuropathy, and analgesic drugs, such as opioids and triptans, can induce hyperalgesic priming, a long-lasting neuroplastic change in nociceptors [2; 5; 7; 12; 24; 34; 51], characterized by marked prolongation of prostaglandin E₂ (PGE₂)-induced hyperalgesia and nociceptor sensitization [19; 20; 26; 47; 50; 54; 55; 68]. Two distinct types of priming have been identified. Type I, produced by activation of protein kinase C epsilon (PKCε) and maintained by protein translation in the terminals of nociceptors [2; 24; 51], is induced by carrageenan, interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), sumatriptan, intradermal fentanyl, and systemic morphine [2; 6; 8; 12; 16; 21; 24; 25; 50; 54; 55]. Type II, produced by repeated exposure to DAMGO, a selective μ-opioid receptor (MOR) agonist, and intrathecal fentanyl [7; 12], is maintained by the activity of two kinases, Src and mitogen-activated protein kinase (MAPK) [7; 12].

In the present experiments, we utilized a model of unpredictable stress produced by non-habituating sound, with properties similar to PTSD. Previous studies have shown that sound stress (SS) activates the sympathoadrenal stress axis [63] and increases plasma epinephrine levels [30], leading to enhanced mechanical pain [38]. This model could also be reasonably related to the types of noise experienced by construction workers, especially in terms of amplitude and unpredictability [64]. Given the established link between stress and chronic pain, we aimed to investigate whether unpredictable sound stress induces hyperalgesic priming and whether it is Type I and/or Type II.

Materials & Method

Animals

Experiments were performed on adult male Sprague–Dawley rats, supplied by Charles River Laboratories (Hollister, CA, USA). Given the large number of experiments (and rats) required to establish whether unpredictable sound stress induces hyperalgesic priming and its underlying mechanisms, and the greater impact of sound stress in male-based occupations, we elected to perform experiments in female rats in a subsequent study. Experimental animals were housed three per cage, under a 12-hour light/dark cycle, in a temperature- and humidity-controlled animal care facility at the University of California, San Francisco. Rats had food and water available ad libitum, in their home cage. Experimental protocols, approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California at San Francisco, adhered to the National Institutes of Health Guide for the care and use of laboratory animals. Efforts were made to minimize the number of animals used and their suffering.

Nociceptive threshold testing

Mechanical nociceptive threshold was quantified using an Ugo Basile Analgesymeter^® (Randall-Selitto paw-withdrawal device, Stoelting, Chicago, IL, USA), which applies a linearly increasing mechanical force to the dorsum of a rat’s hind paw [5; 9; 10; 65]. To minimize restraint stress during testing, rats were placed in cylindrical acrylic restrainers designed to provide ventilation and allow hind leg extension from lateral ports, for the testing of mechanical nociceptive threshold. To acclimatize rats to the experimental testing procedure, they were placed in restrainers for 1 hour prior to starting training sessions, daily, for 3 consecutive days, and on subsequent days for 40 minutes prior to experimental manipulations. Nociceptive threshold was defined as the force, in grams, applied by the analgesymeter, at which a rat withdrew its paw. Baseline threshold was defined as the mean of three readings taken before test agents were injected.

Drugs

The following compounds were used in this study: prostaglandin E₂ (PGE₂, a direct-acting hyperalgesic agent that sensitizes nociceptors), PKCεV_1-2 (PKCε-I, a PKCε-specific translocation inhibitor peptide [7; 33; 39], cordycepin 5′-triphosphate sodium salt (a protein translation inhibitor; [23; 24]), SU6656 (a Src family kinase inhibitor, [9; 12]), and U0126 (a MAPK/ERK inhibitor; [9; 12]), all of which were purchased from Sigma-Aldrich (St. Louis, MO, USA); and H-89 dihydrochloride (an inhibitor of PKA, [7]), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

The stock solution of PGE₂ (1 μg/μL) was prepared in 10% ethanol with additional dilutions made with physiological saline (0.9% NaCl), yielding a final ethanol concentration <1%. PKCε inhibitor was dissolved in saline. H-89, SU6656, and U0126 were dissolved in 100% DMSO (Sigma-Aldrich) and further diluted in saline containing 2% Tween 80 (Sigma-Aldrich). The final concentration of DMSO and Tween 80 was ~2%. Importantly, control experiments have shown that the intradermal injection of the final concentration of ethanol used to prepare the solution of PGE₂ (2%) alone had no effect on the mechanical threshold [13]; 2% DMSO and Tween 80, used to dissolve H-89, SU6656, and U0126 also did not affect nociceptive threshold [13].

Intradermal administration of test agents was performed at the site of nociceptive threshold testing, on the dorsum of the hindpaw, using a 30-gauge hypodermic needle adapted to a 50 μL Hamilton (Reno, NV, USA) syringe by PE-10 polyethylene tubing (Becton Dickinson; Franklin Lakes, NJ, USA). The combination of SU6656 and U0126 was diluted to a concentration of 1 μg/2 μL each and the combination injected by adding 2 μL into a syringe separated by an air bubble to avoid mixing in the syringe. The intradermal administration of PKCεV_1-2 peptide, H-89, and the combination of SU6656 and U0126 were preceded by a hypotonic shock to transiently enhance the permeability of the cell membrane to these agents [17; 18]. This procedure involved administering 1 μL of distilled water, separated from the drug by an air bubble to prevent mixing in the syringe; this volume of distilled water, alone, does not affect the mechanical nociceptive threshold [13].

Components of Hyperalgesic Priming

Hyperalgesic priming involves three phases: (1) induction, (2) expression, and (3) maintenance, the latter, underlies the long-term persistence of this neuroplastic state, contributing to preclinical models of chronic pain. To interfere in each of the three components, inhibitors are administered at different time points as schematically demonstrated in Figure 1. To determine if an inhibitor prevents induction of priming, it is administered before the inducer (e.g., carrageenan, IL-6, TNF-α, morphine, DAMGO, fentanyl, sumatriptan, stress, [2; 5-9; 12; 16; 21; 24; 25; 29; 50; 54; 55]). In this study, no inhibitors were administered to the rats prior to exposure to sound stress. If priming is already present and an inhibitor blocks the prolongation of PGE₂-induced hyperalgesia, measured at the 4^th hour after administration of PGE₂, that drug inhibited the expression of priming. And, if PGE₂ is administered 15-30 days after the inhibitor, and the prolongation of PGE₂ hyperalgesia at the 4^th hour is again absent, that inhibitor has reversed the maintenance of priming.

Figure 1. The three phases of hyperalgesic priming: induction, expression, and maintenance.

To disrupt each phase, inhibitors are given at different times, as illustrated in this figure. Induction: To determine if an inhibitor blocks the induction of priming, it should be administered before the priming agent (e.g., carrageenan, IL-6, TNF-α, morphine, DAMGO, fentanyl, sumatriptan or stress). In this study, no inhibitors were administered to rats prior to sound-stress exposure. Expression: If priming is already present, and an inhibitor blocks the prolongation of PGE₂-induced hyperalgesia at the 4^th hour, the inhibitor has affected the expression of priming. Maintenance: If PGE₂ is reinjected 15 to 30 days after the inhibitor is administered, and the prolongation of PGE₂ hyperalgesia, measured at the 4^th hour after PGE₂ remains inhibited, the inhibitor has reversed the maintenance of priming. Schematic created with BioRender.com.

Unpredictable Sound Stress

Exposure to unpredictable sound stress (SS) occurred over 4 days, as described previously [4; 36; 38; 60; 61; 63]. Rats were housed in groups of three in the housing room, and the same group was transferred to a 30 × 40 × 24 cm wire-mesh cage, 25 cm from a loudspeaker, inside a 56 × 56 × 72 cm sound-insulated box (fabricated by the UCSF machine shop). The box was lead-lined and equipped with a hinged, latched door and internal acoustic panels to attenuate external noise, and contained a loudspeaker mounted on the inner wall connected to a CD player and amplifier positioned on top of the chamber for delivery of acoustic stimuli. Sound pulses were emitted as pure tones, at three frequencies (11, 15, and 19 kHz) with amplitudes varying from 20 to 110 dB, independently for each frequency. The sound stress protocol was initiated after placing rats in the wire mesh cage and terminated 30 min later. Over the 30 min period, a 5 or 10 s tone was presented every minute, at random times. Exposure to the sound stressor occurred on days 1, 3, and 4. Sham stressed animals were placed in the chamber used for sound stress for 30 min at the same time points, but without exposure to the sound stimulus. After each session, rats were returned to their original cage and position in the housing room. Cages in the housing room were arranged side by side by treatment group, separated by shelves on a non-ventilated rack. Eighteen days after the first exposure to sound stress or sham, over which time neuroplastic changes induced by sound stress are established [36; 37; 61], animals were used for behavioral experiments. To minimize experimenter bias, the individual conducting the behavioral experiments (D.A.) was blinded to experimental group.

Data Analysis

Data are presented as mean ± SEM of n independent observations. Statistical comparisons were made using GraphPad Prism 10.5 statistical software (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 is considered statistically significant. In the experiments, the dependent variable is the change in mechanical paw-withdrawal threshold, expressed in grams (g) and/or as percentage change from baseline. As specified in figure legends, Student's t test or two-way repeated-measures ANOVA followed by Bonferroni's post hoc test was performed to compare the magnitude of PGE₂-induced hyperalgesia. Table 1 demonstrates that exposure of rats to either sham sound stress or sound stress does not alter the mechanical nociceptive threshold when comparing the measurements taken before (day 0) and after (day 18) the exposure.

Table 1.

Mechanical nociceptive threshold, in grams, before (day 0) and after (day 18) sound stress or sham exposure.

	Group	Average paw-withdrawal threshold before (day 0)	Average paw-withdrawal threshold after (day 18)	Statistics (paired Student’s t test)	n
Figure 2.	Sham	118.57 ± 3.88 g	120.67 ± 3.92g	p = 0.1725, t(5) = 1.433	6
	Sound Stress	115.33 ± 1.61 g	119.31 ± 1.84 g	p = 0.4838, t(5) = 0.7559	6
Figure 3.	Sham, vehicle	119.67 ± 3.88 g	121.33 ± 3.92 g	p = 0.1852, t(5) = 1.536	6
	Sham, PKCε inhibitor	119.13 ± 4.46 g	115.33 ± 2.81 g	p = 0.1019, t(5) = 2.000	6
	Sound Stress, vehicle	120.00 ± 2.83 g	118.67 ± 1.91 g	p = 0.7556, t(5) = 0.3288	6
	Sound Stress, PKCε inhibitor	119.33 ± 2.46 g	120.33 ± 2.50 g	p = 0.7015, t(5) = 0.4060	6
Figure 4.	Sham, vehicle	127.00 ± 2.11 g	126.00 ± 2.63 g	p = 0.5177, t(5) = 0.6956	6
	Sham, PKA inhibitor	122.00 ± 2.73 g	120.00 ± 2.00 g	p = 0.2752, t(5) = 1.225	6
	Sound Stress, vehicle	120.10 ± 3.27 g	123.67 ± 2.98 g	p = 0.0822, t(5) = 2.169	6
	Sound Stress, PKA inhibitor	117.67 ± 2.65 g	119.67 ± 2.39 g	p = 0.3632, t(5) = 1.000	6
Figure 5.	Sham, vehicle	131.67 ± 3.48 g	127.67 ± 1.75 g	p = 0.1357, t(5) = 1.777	6
	Sham, cordycepin	132.67 ± 5.48 g	130.67 ± 5.33 g	p = 0.5301, t(5) = 0.6742	6
	Sound Stress, vehicle	116.00 ± 2.07 g	114.67 ± 1.84 g	p = 0.7089, t(5) = 0.3953	6
	Sound Stress, cordycepin	116.33 ± 1.20 g	119.33 ± 1.84 g	p = 0.1647, t(5) = 1.627	6
Figure 6.	Sham, vehicle	120.00 ± 2.83 g	120.33 ± 2.70 g	p = 0.9311, t(5) = 0.0909	6
	Sham, kinase inhibitors	119.33 ± 2.46 g	116.33 ± 3.52 g	p = 0.2956, t(5) = 1.168	6
	Sound Stress, vehicle	124.33 ± 4.66 g	125.33 ± 3.64 g	p = 0.6560, t(5) = 0.4732	6
	Sound Stress, kinase inhibitors	124.66 ± 5.48 g	126.33 ± 4.80 g	p = 0.4186, t(5) = 0.8811	6

Data Availability

Upon a reasonable request, the data generated during the current study are available from the corresponding author.

Results

Unpredictable sounds stress induces hyperalgesic priming

Hyperalgesic priming, characterized by marked prolongation of PGE₂-induced hyperalgesia, has served as a preclinical model of the transition from acute to chronic pain. Because unpredictable stress is an important risk factor for the development of chronic pain (e.g., PTSD [58], FMS [27], and IBS [56]) we investigated whether it induces hyperalgesic priming. Given that activation of neuroendocrine stress axes and neuroplastic changes in primary afferent nociceptors are established 18 days after the first exposure to sound stress [4; 36; 37], we evaluated for the presence of hyperalgesic priming at this time point. Both sound-stressed and sham-stressed rats received an intradermal injection of PGE₂ (100 ng/5 μL, i.d.), and mechanical nociceptive threshold evaluated 30 min and 4 hours later. Hyperalgesia was present in both groups when measured 30 min after administration of PGE₂ (Figure 2A,B). However, the prolongation of PGE₂-induced hyperalgesia, measured at the 4^th hour, was present only in the sound-stressed group (Figure 2A,B), indicating that unpredictable sound stress induces hyperalgesic priming.

Figure 2. Unpredictable sound stress induces prolongation of PGE₂ hyperalgesia.

Exposure to unpredictable sound stress occurred on days 1, 3 and 4. Eighteen days after the first exposure to sound stress or sham, PGE₂ was administered intradermally (i.d., 100 ng/5 μL, i.d.), and mechanical nociceptive threshold evaluated 30 min (0.5 h) and 4 hours later. In both groups, PGE₂ induced hyperalgesia at 0.5 h. However, only in the sound stress group did PGE₂ hyperalgesia persist at the 4^th hour, compatible with the presence of hyperalgesic priming. A. Data is presented in mechanical threshold (grams), mean±SEM. Two-way repeated-measures ANOVA, time x sound stress interaction, F_(10,30) = 7.202, p< 0.0001; Sound stress, F_(1,10) = 8.685, p = 0.0146; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham vs. sound stress at the 4^th hour after PGE₂). B. The same data set is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × sound stress interaction, F_(10,20) = 1.988, p = 0.0916; Sound stress, F_(1,10) = 21.9, p = 0.0009; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham vs. sound stress at the 4^th hour after PGE₂). N = 6 rats per group.

Prolongation of PGE₂-induced hyperalgesia in SS primed rats (expression) is not PKCε dependent

Currently, two types of priming have been established; referred to as Type I and Type II. We first focused on a potential role of Type I priming, the type induced by agents such as carrageenan, IL-6, TNF-α, and systemic morphine [2; 16; 24; 25; 54], in SS-induced priming. The prolongation of PGE₂ hyperalgesia (i.e., expression of priming) in Type I priming is mediated by PKCε and maintained by peripheral protein translation [2; 51]. Therefore, we tested whether the prolongation of PGE₂-induced hyperalgesia by unpredictable sound stress is PKCε dependent. Eighteen days after the first exposure to sound stress, rats were treated intradermally with a PKCε inhibitor (PKCεV_1-2 peptide, 1 μg/5 μL, i.d.) or its vehicle (5 μL, i.d.), followed 10 min later by PGE₂ (100 ng/5 μL, i.d.), administered at the same site on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated 30 minutes and 4 hours after PGE₂ administration. PGE₂-induced hyperalgesia was observed in both the PKCε inhibitor-treated group and the vehicle control at 30 minutes and 4 hours (Figure 3A,B), indicating that the expression of SS-induced priming is not PKCε dependent and SS-induced priming is not Type I.

Figure 3. Sound stress-induced hyperalgesic priming is not PKCε dependent.

Sound stress or sham sound stress exposure occurred on days 1, 3 and 4. Eighteen days after the first exposure to sound stress or sham, rats were treated with PKCε inhibitor (PKCεV_1-2 peptide, 1 μg/5 μL, i.d.) or its vehicle (saline, 5 μL, i.d.) intradermally on the dorsum of the hindpaw, followed, 10 min later, by PGE₂ (100 ng/5 μL, i.d.), administered at the same site. Mechanical nociceptive threshold was evaluated at this site, before and 18 days after the first exposure to sound stress or sham, and then 30 min and 4 hours after PGE₂ administration. In SS-exposed rats neither PGE₂-induced hyperalgesia at 30 min or its prolongation at the 4^th hour, were affected in rats treated with the PKCε inhibitor or its vehicle. These data indicate that sound stress-induced priming is not PKCε-dependent.

A. Mechanical threshold (grams) data is presented as mean ± SEM. Two-way repeated-measures ANOVA, time × PKCε inhibitor interaction, F_(20,60) = 3.751, p < 0.0001; Sound stress, PKCε inhibitor interaction, F_(3,20) = 7.052, p= 0.0020; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham vs. sound stress at the 4^th hour after PGE₂), ns, p > 0.9999 (SS, vehicle vs. SS, PKCε inhibitor, at the 4^th hour after PGE₂). B. The same data set is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × PKCε inhibitor interaction, F_(20,40) = 1.228, p = 0.2827; Sound stress, PKCε inhibitor interaction, F_(3,20) = 22.43, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham vs. sound stress at the 4^th hour after PGE₂), and ns, p > 0.9999 (SS, vehicle vs. SS, PKCε inhibitor, at the 4^th hour after PGE₂). N = 6 rats per group.

Maintenance of SS-induced hyperalgesic priming is not dependent on peripheral protein translation

Type I priming is maintained by peripheral protein translation, as evidenced by the finding that the protein translation inhibitor cordycepin permanently reverses Type I priming [8; 21; 24]. To evaluate if sound stress-induced priming is maintained by peripheral protein translation, 18 days after rats were exposed to sound stress or sham, we treated the hindpaws of rats intradermally with either a protein translation inhibitor (cordycepin; 1 μg/5 μL, i.d.) or its vehicle (saline; 5 μL, i.d.) followed 10 min later by PGE₂ (100 ng/5 μL, i.d.), administered at the same site, on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated before (day 0) and 18 days after the first exposure to sound stress or sham, and as well as 30 min and 4 hours after intradermal PGE₂. In sound-stressed rats treated with vehicle or cordycepin, hyperalgesia at both 30 min and 4 hours after PGE₂ remained unchanged (Figure 4A,B). These findings support the suggestion that hyperalgesic priming induced by sound stress is not Type I.

Figure 4. SS-induced hyperalgesic priming is not dependent on peripheral protein translation.

Sound stress or sham exposure occurred on days 1, 3 and 4. Eighteen days after the first exposure to sound stress or sham, rats were treated with a protein translation inhibitor, cordycepin (1 μg/5 μL, i.d.), or its vehicle (saline, 5 μL, i.d.) administered intradermally on the dorsum of the hindpaw, followed, 10 min later, by PGE₂ (100 ng/5 μL, i.d.) administered at the same site. Mechanical nociceptive threshold was evaluated at this site, before and 18 days after the first exposure to sound stress, and then 30 min and 4 hours after PGE₂ administration. In SS-exposed rats neither PGE₂-induced hyperalgesia at 30 min or its prolongation at the 4^th hour, were affected in rats treated with cordycepin or its vehicle. These data indicate that sound stress-induced priming does not rely on peripheral protein translation and is therefore, not Type I priming.

A. Mechanical threshold (grams) data is presented as mean ± SEM. Two-way repeated-measures ANOVA, time × cordycepin interaction, F_(20,60) = 8.285, p < 0.0001; Sound stress, cordycepin interaction, F_(3,20) = 23.14, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham vs. sound stress at the 4^th hour after PGE₂), ns, p > 0.9999 (SS, vehicle vs. SS, cordycepin, at the 4^th hour after PGE₂). B. The same data set is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × cordycepin interaction, F_(20,40) = 0.5670, p = 0.9126; Sound stress, cordycepin interaction, F_(3,20) = 39.19, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham vs. sound stress at the 4^th hour after PGE₂), and ns, p > 0.9999 (SS, vehicle vs. SS, cordycepin, at the 4^th hour after PGE₂). N = 6 rats per group.

Expression and maintenance of SS-induced hyperalgesic priming are PKA dependent

We have previously demonstrated that repeated exposure to DAMGO, a selective μ-opioid receptor (MOR) agonist, induces priming that is maintained by the combined activation of Src and MAPK at the nociceptor peripheral terminal, rather than by peripheral protein translation, which we named Type II priming [7]. The expression of type II priming is PKA- rather than PKCε-dependent as observed in Type I priming [7]. Therefore, we investigated whether PKA, which is also involved in the acute response to PGE₂ hyperalgesia, at 30-min, in Type I and II primed and naïve/control rats [1; 7], plays a role in the expression of SS-induced hyperalgesic priming. Eighteen days after the first exposure to sound stress or sham, rats were treated intradermally with a PKA inhibitor (H-89; 1 μg/5 μL, i.d.) or its vehicle (5 μL, i.d.), followed 10 min later by PGE₂ (100 ng/5 μL, i.d.) administered at the same site on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated at the same site, 30 minutes and 4 hours after PGE₂ administration. Measured 30 min after PGE₂, hyperalgesia was markedly inhibited in the H-89-treated sham and SS groups (Figure 5A,B). And, in SS-exposed rats, the prolongation of PGE₂ hyperalgesia at the 4^th hour was also inhibited by the PKA inhibitor (Figure 5A,B). To determine whether maintenance of SS-induced priming is also PKA-dependent, thirty days after the administration of the PKA inhibitor (H-89), rats were again tested for priming, by again receiving intradermal PGE₂ (100 ng/5 μL, i.d.) and mechanical nociceptive threshold evaluated 30 min and 4 hours later. Importantly, unlike in Type II priming, whose maintenance is mediated by Src and MAPK, the prolongation of PGE₂-induced hyperalgesia at the 4^th hour was still markedly attenuated in the PKA inhibitor-treated group (Figure 5B, right panel), indicating that maintenance, as well as the expression, of SS-induced hyperalgesic priming is PKA-dependent.

Figure 5. Expression and maintenance of sound stress-induced priming are PKA dependent.

Sound stress or sham exposure occurred on days 1, 3 and 4. Eighteen days after the first exposure to sound stress or sham, rats were treated intradermally (i.d.) with PKA inhibitor (H-89, 1 μg/5 μL) or its vehicle (5 μL), on the dorsum of the hindpaw, followed, 10 min later, by PGE₂ (100 ng/5 μL, i.d.), administered at the same site. Mechanical nociceptive threshold was measured before the first exposure to sound stress (day 0), 18 days after the first exposure, and then 30 minutes and 4 hours following PGE₂ administration. The PKA inhibitor significantly reduced PGE₂-induced hyperalgesia at 30 minutes in both sham- and sound-stressed rats (A,B), compared to vehicle-treated groups. In the sound-stressed group, the PKA inhibitor also inhibited the prolongation of PGE₂-induced hyperalgesia at 4 hours. When rats were retested for priming 30 days after PKA inhibitor administration, the prolongation of PGE₂ (100 ng/5 μL, i.d.)-induced hyperalgesia was still inhibited in the sound-stressed group. These findings indicate that the expression and maintenance of sound stress-induced priming is PKA-dependent.

A. Mechanical threshold (grams) data is presented as mean ± SEM. Two-way repeated-measures ANOVA, time × PKA inhibitor interaction, F_(20,60) = 3.054, p = 0.0004; Sound stress, PKA inhibitor interaction, F_(3,20) = 16.47, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham, PKA inhibitor vs. sound stress, PKA inhibitor, at 30 min and 4^th hour after PGE₂). B. The same data set is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × PKA inhibitor interaction, F_(20,40) = 1.716, p = 0.0721; Sound stress, PKA inhibitor interaction, F_(3,20) = 61.48, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (sham, PKA inhibitor vs. sound stress, PKA inhibitor, at 30 min and 4^th hour after PGE₂). Third days after PKA inhibitor: Data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × PKA inhibitor interaction, F_(10,20) = 1.093, p = 0.4122; Sound stress, PKA inhibitor interaction, F_(1,10) = 40.44, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (Sound stress, vehicle vs. sound stress, PKA inhibitor, at the 4^th hour after PGE₂). N = 6 rats per group.

Effect of inhibitors for maintenance of Type II hyperalgesic priming on SS-induced priming

The combined administration of inhibitors of Src and MAPK, to the nociceptor peripheral terminal, reverses Type II hyperalgesic priming [9; 12]. Therefore, to evaluate whether SS-induced priming is Type II, 18 days after the first sound stress or sham exposure, hindpaws were treated with the combination of a Src (SU6656, 1 μg/2 μL, i.d.) and a MAPK (U0126, 1 μg/2 μL, i.d.) inhibitor, or their vehicle (saline containing 2% of DMSO and Tween 80; 5 μL, i.d.), followed 10 min later by PGE₂ (100 ng/5 μL, i.d.) administered at the same site on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated before (day 0) and 18 days after the first sound stress or sham exposure, and as well as 30 min and 4 hours after intradermal PGE₂. In the sham- and sound-stressed groups treated with the combination of a Src and MAPK inhibitor, PGE₂-induced hyperalgesia at 30 min was slightly inhibited (Figure 6A,B) and markedly inhibited at the 4^th hour (Figure 6A,B). These findings indicate that Src and MAPK play a role in the expression of SS-induced priming. To evaluate their role in the maintenance of SS-induced priming, PGE₂ (100 ng/5 μL, i.d.) was reinjected 17 days after the administration of the inhibitors. The prolongation of PGE₂-induced hyperalgesia remained slightly, but significantly, inhibited in the sound-stressed group that received the combination of a Src and a MAPK inhibitor (Figure 6B, right panel); however, inhibition was substantially less than the near complete reversal observed in Type II priming [9; 12]. These findings suggest that while the maintenance of SS-induced priming is strongly dependent on PKA, there may be a transient contribution of Src and MAPK.

Figure 6. Effect of inhibitors of the expression and maintenance of Type II priming on sound stress-induced priming.

Exposure to sound stress or sham occurred on days 1, 3 and 4. Eighteen days after the first exposure to sound stress or sham, rats were treated intradermally (i.d.) with a Src (SU6656, 1 μg/2 μL, i.d.) plus a MAPK (U0126, 1 μg/2 μL, i.d.) inhibitor, or their vehicle (5 μL), on the dorsum of the hindpaw, followed, 10 min later, by PGE₂ (100 ng/5 μL, i.d.), administered at the same site. Mechanical nociceptive threshold was evaluated at this site, before (day 0) and 18 days after the first exposure to sound stress, and then 30 min and 4 hours after PGE₂ administration. The combination of Src and MAPK inhibitors slightly reduced PGE₂-induced hyperalgesia at 30 minutes in both sham-stressed and sound-stressed rats (A,B), compared to vehicle-treated groups. In the sound-stressed group, the combination of Src and MAPK inhibitors markedly attenuated the prolongation of PGE₂-induced hyperalgesia at 4 hours. These findings indicate that the expression of SS-induced priming is Src- and MAPK-dependent. When rats were retested for priming with PGE₂ (100 ng/5 μL, i.d.) 17 days after the combined administration of Src and MAPK inhibitors, the prolongation of PGE₂-induced hyperalgesia in the sound-stressed group remained only slightly inhibited, unlike in Type II priming (B).

A. Mechanical threshold (grams) data is presented as mean ± SEM. Two-way repeated-measures ANOVA, time × Src and MAPK inhibitors interaction, F_(20,60) = 6.474, p< 0.0001; Sound stress, Src and MAPK inhibitors interaction, F_(3,20) = 3.326, p = 0.0405; Bonferroni's multiple post hoc comparisons test: *p = 0.0127, *p = 0.0340, ***p = 0.0001 (sham, vehicle × Src and MAPK inhibitors; Sound stress, vehicle × Src and MAPK inhibitors, at 30 min and 4^th hour after PGE₂). B. The same data set is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × Src and MAPK inhibitors interaction, F_(20,40) = 0.5566, p = 0.9194; Sound stress, Src and MAPK inhibitors interaction, F_(3,20) = 40.03, p < 0.0001; Bonferroni's multiple post hoc comparisons test: *p = 0.0421, *p = 0.0128, ***p = 0.0004 (sham, vehicle × Src and MAPK inhibitors vs. sound stress, vehicle × Src and MAPK inhibitors, at 30 min and 4^th hour after PGE₂). Seventeen days after Src and MAPK inhibitors: Data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × Src and MAPK inhibitors, F_(10,20) = 0.7720, p = 0.6538; Sound stress, Src and MAPK inhibitors, F_(1,10) = 5.314, p = 0.0439; Bonferroni's multiple post hoc comparisons test: *p = 0.0299 (Sound stress, vehicle vs. Sound stress, Src and MAPK inhibitors, at the 4^th hour after PGE₂). N = 6 rats per group.

Discussion

Unpredictable stress plays an important role in the pathophysiology of chronic pain, as part of a vicious cycle whereby stress triggers or worsens pain through enhancing muscle tension, inflammation (neuroimmune), and neural activity, and neuroendocrine dysregulation, while chronic pain enhances activity in neuroendocrine stress axes, impacting mental health and overall quality of life. This reciprocal reinforcement leads to increased sensitivity to noxious stimuli, reduced pain tolerance, and a heightened stress response enhancing chronic pain. Rats subjected to the early-life stress induced by neonatal limited bedding (NLB), had increased neuroendocrine stress axis responsiveness [28; 31; 46], developed mechanical hyperalgesia [3; 29], and PKCε-dependent nociceptor plasticity [29]. Additionally, unpredictable sound stress exacerbates the pain associated with chemotherapy-induced peripheral neuropathy (CIPN) produced by the administration of oxaliplatin [61] and paclitaxel [22]. Here, for the first time, we have demonstrated that unpredictable stress induces hyperalgesic priming, a form of peripheral nociceptor plasticity identified by the prolongation of PGE₂-induced hyperalgesia [19; 20; 26; 47; 50; 54; 55; 68].

Unpredictable SS-induced nociceptor plasticity likely results from both inflammatory processes and activation of neuroendocrine stress axes. The effect of stress on inflammatory responses is influenced by the type and duration of the stressor. The sympathoadrenal stress axis modulates inflammation, predominantly through epinephrine release, which acts on adrenergic receptors on leukocytes and nociceptors [36; 57; 66]. β₂-adrenergic receptor agonists stimulate generation of pronociceptive molecules such as reactive oxygen species [15; 48] and interleukin-8, in macrophages [35], while α₁-adrenergic receptors increase leukocyte migration and enhance the inflammatory response [32]. Previous work demonstrated that PGE₂-induced hyperalgesia, assessed 24 hours after the last of four SS exposures did not differ from sham controls [36]. However, when measured 14 days after the last SS exposure, prolonged PGE₂-induced hyperalgesia was observed [36]. Notably, in that study mechanical nociceptive threshold was only assessed at 20 minutes post-injection of PGE₂, which does not evaluate for the presence of priming (prolongation of PGE₂-induced hyperalgesia at 4+ hours). Additionally, that study used a protocol involving cumulatively increasing doses of PGE₂ (0.1–1000 ng), administered at 25-minute intervals [36].

For more than two decades [2; 51], hyperalgesic priming has been studied as a model of the transition from acute to chronic pain. Priming encompasses three phases: induction, expression, and maintenance (Figure 1). To date, two mechanistically distinct types of priming have been identified, referred to as Type I and Type II. Type I can be induced by carrageenan, IL-6, TNF-α, sumatriptan, fentanyl (at the nociceptor peripheral terminal), and systemic morphine [2; 6; 8; 12; 16; 21; 24; 25; 50; 54; 55]. The induction and expression of Type I priming depend on the activation of PKCε, while its maintenance relies on protein translation in the terminals of the nociceptor [2; 24; 51]. In the present experiments, we demonstrated that unpredictable SS induces priming whose expression is not PKCε dependent and whose maintenance is not peripheral protein translation dependent. These findings indicate that SS-induced priming is not Type I.

Type II priming, which can be induced by repeated administration of DAMGO (at the nociceptor peripheral terminal) and by fentanyl (at the nociceptor central terminal) [9; 12], is dependent on these opioids acting as MOR agonists [7; 11]. While the expression of Type II priming is PKA dependent [7], in contrast to Type I priming which is PKCε dependent, its maintenance relies on the combined activity of Src and MAPK [9; 12], which contrasts to Type I priming whose maintenance is dependent on protein translation in the terminals of the nociceptor. In the present experiments, we found that the expression of priming induced by unpredictable sound stress is dependent on PKA, which also plays a role in PGE₂-induced hyperalgesia in unprimed (control) animals [2]. That the effect of the PKA inhibitor H-89 does not last more than a few hours [7], excludes the possibility of a prolonged effect of H-89 30 days post its administration, at which time we still observed the reversal of SS-induced priming. We also evaluated whether the combined activity of Src and MAPK, which maintains Type II priming, plays a role in the maintenance of SS-induced priming. Importantly, when PGE₂ was reinjected 17 days after co-administering Src and MAPK inhibitors a small, albeit statistically significant, inhibition of the prolongation of PGE₂ hyperalgesia could still be detected. Given that the effect of Src and MAPK inhibitors decayed markedly 17 days post-administration, in the maintenance protocol, we suggest that SS-induced priming has little if any Type II priming, which would require a prolonged contribution of these two kinases. Importantly, in this regard, combined inhibition of Src and MAPK abolishes the prolongation of PGE₂-induced hyperalgesia in Type II priming induced by DAMGO and fentanyl, when priming was tested 15-30 days after the combined administration of the two kinase inhibitors [9; 12]. Therefore, we suggest that the maintenance of SS-induced priming is almost completely PKA dependent.

Limitations of our initial study of SS-induced hyperalgesic priming focused entirely on the male rat. This decision was informed by the prevalence of men in occupations at high risk for noise pollution, such as urban construction, a field where only 6–12% of workers are women [44; 53]. Thus, future studies in female rats are a future direction of our research on SS-induced hyperalgesic priming. However, it is important to note that our prior findings indicate that unpredictable sound stress exacerbates pain in chemotherapy-induced peripheral neuropathy (CIPN), in both male and female rats treated with oxaliplatin and paclitaxel [22; 60; 61], supporting the suggestion of little if any sex differences in the effect of SS on pain. Additionally, we did not test agents aimed at preventing hyperalgesic priming induced by sound stress, as the primary focus of this study was to validate the presence of priming in rats exposed to unpredictable sound stress, and reversing its maintenance with inhibitors for Type I and II priming. H-89 was initially marketed as a relatively potent inhibitor of PKA; however, subsequent work has demonstrated that it also inhibits multiple other kinases within the AGC family and exerts additional PKA-independent actions on other signaling proteins [40; 41; 45]. Thus, while our findings are consistent with a role for PKA-related signaling, potential contributions of other H-89–sensitive kinases and targets in peripheral tissue cannot be excluded. Together, this work provides a foundation for future studies aimed at identifying effective strategies to reverse the maintenance of hyperalgesic priming, a critical step toward developing disease-modifying treatments for chronic pain.

In conclusion, in the present study we demonstrate that unpredictable sound stress induces a novel form of hyperalgesic priming, which is mediated by mechanisms distinct from those previously characterized for Type I and Type II priming. Unlike Type I priming, SS-induced priming does not depend on PKCε or peripheral protein translation. And, although it shares some features with Type II priming, such as expression that is dependent on PKA, and Src and MAPK signaling, it diverges in its dependence on Src and MAPK pathways for maintenance, which is critical to defining the presence and type of priming that might contribute to a chronic pain syndrome. Our data support the suggestion that unpredictable sound stress engages unique molecular networks supporting long-term nociceptor plasticity. Given that stress and chronic pain reciprocally amplify each other, these findings provide a valuable framework for elucidating how stress-related signaling cascades contribute to vulnerability to persistent pain.

Abbreviations:

ANOVA

Analysis of variance

DMSO

Dimethyl sulfoxide

DAMGO

D-Ala²,N-MePhe⁴,Gly-ol-enkephalin

DRG

Dorsal root ganglion

ERK

Extracellular signal-regulated kinase

FMS

Fibromyalgia syndrome

IBS

Irritable bowel syndrome

i.d

Intradermal(ly)

IL-6

Interleukin-6

MAPK

Mitogen-activated protein kinase

MOR

Mu-opioid receptor

NLB

Neonatal limited bedding

PKA

Protein kinase A

PKCε

Protein kinase C epsilon isoform

PGE₂

Prostaglandin E₂

PTSD

Post-traumatic stress disorder

SEM

Standard error of the mean

SS

Sound stress

TNF-α

Tumor necrosis factor alpha